Cytotoxic CD8+ T cells target citrullinated antigens in rheumatoid arthritis

The immune mechanisms that mediate synovitis and joint destruction in rheumatoid arthritis (RA) remain poorly defined. Although increased levels of CD8+ T cells have been described in RA, their function in pathogenesis remains unclear. Here we perform single cell transcriptome and T cell receptor (TCR) sequencing of CD8+ T cells derived from anti-citrullinated protein antibodies (ACPA)+ RA blood. We identify GZMB+CD8+ subpopulations containing large clonal lineage expansions that express cytotoxic and tissue homing transcriptional programs, while a GZMK+CD8+ memory subpopulation comprises smaller clonal expansions that express effector T cell transcriptional programs. We demonstrate RA citrullinated autoantigens presented by MHC class I activate RA blood-derived GZMB+CD8+ T cells to expand, express cytotoxic mediators, and mediate killing of target cells. We also demonstrate that these clonally expanded GZMB+CD8+ cells are present in RA synovium. These findings suggest that cytotoxic CD8+ T cells targeting citrullinated antigens contribute to synovitis and joint tissue destruction in ACPA+ RA.


March 2021
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Replication GZMK+ cluster. The normalized enrichment score (NES) and adjusted P value for multiple testing were used to define the enriched biological process pathways in either GZMB+ or GZMK+ clusters. The gene sets of gene ontology were obtained from Molecular Signatures Database (v7.5.1) and the GSEA was performed using the fgsea R package.
Here is the version of each R package we used. In this study, sex and gender were not determined as a factor even though most of RA samples were collected from male because we recruited RA patients at VA Palo Alto. We didn't see any difference when we analyzed based on sex difference.
59 RA patients and 30 healthy individuals were included in this study. 45 with ACPA positive and 14 with ACPA negative patients. ACPA positivity was tested by serum ELISA. Reference range for ACPA level; Less than 20 is ACPA Negative. 55 male and 4 female were included. Patient details are listed in Supplementary Table 1.
Among RA patients who came to VA Palo Alto Health Care System or Stanford Hospital, those who voluntarily participated in this study were recruited. RA blood samples were collected under institutional review board (IRB) approved protocols and after written informed consent at VA Palo Alto Health Care System and Stanford University. Healthy samples were obtained through the Stanford Blood Center. Paired RA blood and synovium samples were collected at HSS New York. There are no self-selection bias or other biases that may be present.
All experimental protocols were approved by the institutional review board of Stanford University and VA Palo Alto (IRB 3780).
Single-cell RNA sequencing includes n=18 individuals (ACPA+ RA=12, Healthy control=6). 16,000 CD8+ T cells were recovered by normalization step from all individuals. 10,400 paired TCRab sequences were recovered. The exact sample size of each experiment is described in the relevant Figure legends. Sample sizes were determined based on previous papers, experimental trials or meaningful statistics.
For paired samples, we included 3 ACPA+ RA blood and 4 synovium and 9,360 TCR ab sequences were recovered. Predetermination of sample size calculation was not performed as we included as many sizes as for statistical analysis.
Data with >10% of mitochondrial genes or low (<200) gene counts for single cell RNA seq were excluded from the cell count. Samples with low cell viability were excluded from flow cytometric analysis. were merged using a MNN based batch correction with R package Harmony. We included quantification results if we provide a representative image. All results presented in the manuscript indicate a high data reproducibility.
Randomization was not relevant to this study as this study does not explore group differences. Healthy controls were selected by random participants (no RA symptoms).
Flow cytometric analysis was blinded by assigning unique number of samples. The investigators were not blinded to single cell RNA seq data as ACPA+ RA samples were chosen by ACPA level in serum and a clinical outcome association was performed. Investigators were blinded to group allocation during experiments and analysis of the results.  The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

DLD-1 (ATCC, ATCC® CCL-221)
Cell from commercial source was distributed with certificates of authentication using STR profiling.
Regular mycoplasma testing was done for DLD-1. It tested negative for mycoplasma contamination.
No commonly misidentified cell line n/a n/a n/a n/a For cellular profiling of CD8+ T cells, whole blood was collected and PBMCs isolated using Ficoll-Paque density gradient centrifugation (Sigma Aldrich). Cells were cryopreserved in Recovery Cell Culture Freezing Medium (Thermo Fisher Scientific). Thawed PBMCs were stabilized at 37°C overnight prior to staining with Fixable Viability Stain 510 (BD Bioscience), followed by fluorophore-conjugated antibodies targeting surface molecules in Stain Buffer (BD Bioscience) (Supplementary Table 4). For intracellular staining, PBMCs were re-stimulated with Cell Stimulation Cocktail (Thermo Fisher Scientific), fixed and permeabilized, then stained with intracellular molecule-targeting antibodies.